Constructs used to modify MCAK activity at live centromeres
| Name . | Description . | Purpose . |
|---|---|---|
| GCPB | The centromere-binding domain of CENP-B fused to the C terminus of GFP | A control construct to mark centromeres for live imaging and for use with mRFP-fused constructs |
| RCPB | The centromere-binding domain of CENP-B fused to mRFP | A control construct to mark centromeres in conjunction with GFP constructs or photoactivatable GFP |
| GCPBM | GCPB fused to the N terminus of the neck + motor minimal functional MT-depolymerizing domain of MCAK | Designed to anchor ectopic MT-depolymerizing activity to the centromere |
| RCPBM | RCPB fused to the neck + motor of MCAK | A red version of GCPBM for use with photoactivatable GFP |
| GCPBMhyp | GCPBM with three point mutations in the motor domain that eliminate the MT-depolymerizing activity of MCAK | Controls for any effects from anchoring GCPBM at the centromere. We found this construct to be indistinguishable in appearance and behavior from GCPB |
| RMLM | mRFP fused to the N terminus of motorless MCAK | A dominant-negative construct designed to deplete endogenous MCAK on centromeres |
| RAMFLhyp | mRFP fused to full-length MCAK inactivated with three point mutations in the motor domain and five aurora B phosphorylation sites mutated to alanine | A more potent dominant-negative construct designed to deplete endogenous active MCAK on centromeres |
| Name . | Description . | Purpose . |
|---|---|---|
| GCPB | The centromere-binding domain of CENP-B fused to the C terminus of GFP | A control construct to mark centromeres for live imaging and for use with mRFP-fused constructs |
| RCPB | The centromere-binding domain of CENP-B fused to mRFP | A control construct to mark centromeres in conjunction with GFP constructs or photoactivatable GFP |
| GCPBM | GCPB fused to the N terminus of the neck + motor minimal functional MT-depolymerizing domain of MCAK | Designed to anchor ectopic MT-depolymerizing activity to the centromere |
| RCPBM | RCPB fused to the neck + motor of MCAK | A red version of GCPBM for use with photoactivatable GFP |
| GCPBMhyp | GCPBM with three point mutations in the motor domain that eliminate the MT-depolymerizing activity of MCAK | Controls for any effects from anchoring GCPBM at the centromere. We found this construct to be indistinguishable in appearance and behavior from GCPB |
| RMLM | mRFP fused to the N terminus of motorless MCAK | A dominant-negative construct designed to deplete endogenous MCAK on centromeres |
| RAMFLhyp | mRFP fused to full-length MCAK inactivated with three point mutations in the motor domain and five aurora B phosphorylation sites mutated to alanine | A more potent dominant-negative construct designed to deplete endogenous active MCAK on centromeres |